The broader applicability of our system and method to other liquid-solid reactions involving stepwise synthesis on solid substrates, such as the production of oligonucleotides, oligodeoxynucleotides, oligoribonucleotides, oligosaccharides, proteins, etc., will be apparent to those skilled in the art.
Solid phase peptide synthesis (SPPS) was developed by Merrifield in 1963. The basic procedure is well known. The SPPS method typically begins with a polymer gel, such as a partially chlorinated polystyrene cross-linked with divinyl benzene. The C-terminal of a protected amino acid is initially bound to the resin, for example, by means of a benzyl ester of the amino acid. Other binding agents may of course be used. The peptide is synthesized in sequence from the C-terminus with protected amino acids. The amino groups and all reactive side-chain functional groups of the amino acids must be protected by stable blocking groups in order to prevent undesirable side reactions. The blocking groups are selected such that the amino group may be deprotected without disturbing the side chain protecting groups or the link between the C-terminus and the resin. The amino group may be protected by, for example, boc (t-butoxy carbonyl) or fmoc (9-fluorenyl methoxy carbonyl) groups.
The peptide synthesis is typically conducted by the following procedure: The N-terminus of the resin-bound peptide (protected by boc) is deblocked in a solution of trifluoroacetic acid (TFA) in dichloromethane (DCM), for example. The next amino acid in the sequence is coupled to the resin-bound peptide with a coupling agent such as dicyclohexylcarbodiimide (DCC) in a solution of DCM and dimethyl formamide (DMF), for example. An activating agent such as 1-hydroxybenzotrazole (HOBt) may be used to improve the rate and selectivity of the coupling reaction and to decrease racemization. The unreacted amino acid, reagents, and by-products are removed from the resin by washing and filtration. The washing and filtration process is then repeated. The N-terminus of the peptide is then de-blocked, another peptide is added to the chain, the system is then washed and filtered, etc. The process is repeated until all the desired amino acids have been added to the peptide chain in the desired order. The remaining blocking groups are then removed from the peptide, the peptide is cleaved from the resin, and the peptide is collected.
SPPS originally was performed, and often still is performed, in shaken or stirred flasks in which the resin is dispersed. To suspend and mix the resin in a fluid phase, several times the amount of liquid which the resin will absorb or hold is required for the system. Thus, if the resin will hold 10 ml of liquid, 50-100 ml of liquid will be required to disperse and suspend the resin. The increased amount of liquid leads to the use of dilute solutions (typically a 150 mmol amino acid/liter of solution) to minimize costs, since the amino acids are expensive. With dilute solutions, it is difficult to obtain high concentrations of amino acids and hence fast chemical reactions between the amino acids and the growing chain. Further, when the resin is washed and filtered (in a flask) via batch dilution, it is virtually impossible, in a limited number of batch washes, to remove from the resin all the DCM and TFA used. As a result, the contact time of the peptide chain with the DCM and TFA cannot be accurately controlled.
A number of design issues in the reactor development are unique to peptide synthesis. The resin used in solid-phase peptide synthesis is usually a gel resin with a low degree of cross-linking. These resins swell in certain solvents (such as DCM), and shrink in other solvents (such as methanol). The resin volume tends also to increase with the growth of the peptide chain length. In addition, these resins tend to be fairly soft in nature and, thus, are sensitive to physical attrition. A reactor accommodating these characteristics is desired. The amount of exposure time between the peptide-resin and the solvents and reactants used in the synthesis also may be very important. Deprotection of the resin-bound peptide must be complete in order to obtain the highest yield, but the resulting carbocations must not remain in contact with the peptide because of undesirable side-reactions that may occur. Unfortunately, the time required to drain the solution from a resin slurry suspension increases with the depth of the resin bed formed during filtration. Consequently, exposure time of resin-bound peptide to carbocations increases as bed depth increases. As a result, the filtration times for kilogram-scale reactions are far longer than those encountered in bench-scale reactions, so the risk of damage to the peptides due to reactions with carbocations increases with the batch size. Larger scale reactors must therefore be designed in such a way as to mirniize the filtration time. Several solutions to this problem can be selected by the manner of reactor design and through resin particle properties permitted by the reactor design.
In order for a chemical reaction to occur, reactant species must be present at the reaction site within the beads. The reactions involved in the coupling step may be limited by the rate at which reactants (DCC, and the intermediates from the bulk liquid phase of the coupling reactor) permeate through the surface and pores of the resin to internal reaction sites. As is known, the resin, which is typically a polystyrene resin, is effectively formed of porous spheres. The initial amino acid of the peptide chain to be formed is bonded to internal sites throughout the resin matrix. The peptide chain then begins to grow at these sites as the reactant species arrive at the sites.
The rate of mass transfer of reactants from the bulk liquid to the sites within the resin is proportional to the concentration gradient multiplied by the diffusion coefficient of the solute through the solvent (Fick's Law), but it is convenient to think of mass transfer in terms of resistances. Resistance to mass transfer in heterogeneous, solid-liquid processes is evident at two places: (1) across the stagnant liquid film at the surface of the resin particle; and (2) within the pores of the resin particle. Increasing the fluid velocity relative to the resin reduces the film resistance. To reduce resistance to mass transfer within the resin, the resin beads are preferably small and in a gel state (i.e., 5-100.mu.). The use of more gelatinous resins may permit loadings that exceed 1 mmol of active sites (or peptide chains)/gm resin and approach 2-4 mmol/gm at high yields and conversions. The use of small resin particles is also expected to facilitate better, faster and more complete washing of the resin, because it will be easier to get effective matrix exchange with the wash solvent. Further, the resin beads preferably have a small amount of cross-linking to open up their pores. However, some cross-linking is needed in the resin to give shape and add strength to the resin beads. Resin beads with cross-linking of between 0.2% and 1.0% should be sufficient.
In the synthesis method where amino acids are activated by dicyclohexylcarbodiimide (DCC), the resistance to diffusion into the resin core may be greatly increased by the formation of dicyclohexylurea (DCU), which is essentially insoluble in the solution of 50% DMF in DCM used during the coupling reaction. A shell of insoluble DCU may form on the resin. This deposit of DCU hinders reactants and washes from transferring between the surface and the core of the resin bead. In extreme cases, one may find that diffusion from the bulk liquid phase to the free amines is impossible, and that the only boc-amino acid available for coupling is the material initially present within the resin before the addition of DCC.
The diffusion of reactants into the resin may also be hindered by the formation of the peptide chains themselves. It is generally known that heterogeneous reactions in porous particles typically grow radially inward relative to the outer surface of the particle. It is reasonable to apply this to solid phase synthesis. The bead thus likely comprises a growing shell of reacted peptide and a shrinking unreacted core. If the rate of reaction between the free amine and the activated intermediate is fast relative to the diffusion rate through the resin pores, a shell of reacted peptide, and possibly DCU, will grow radially inward from the surface of the particle. The reaction will take place upon the surface of a shrining core within the resin bead. Reactants must therefore diffuse through an increasingly thick layer of peptide and DCU in order to reach the unreacted core of the resin beads.
One solution to the precipitation of DCU is a change in the chemistry of the reaction system. Resistance to diffusion through DCU-obstructed pores may be reduced or eliminated by forming the symmetric anhydride, o-acyl isourea, and HOBt active ester in a separate reactor (i.e., preactivating the amino acid) and filtering the DCU from the solution prior to introduction into a main reactor where the synthesis is conducted. Similar benefits may be realized if a different coupling agent (such as DIC) is used which does not form an insoluble product during the coupling reaction.
In addition to reducing obstruction by the formation of insoluble compounds such as DCU, pore diffusion limitations also may be reduced by using smaller particles. As a result of decreasing mass transfer resistance, lower concentrations of reactants will be required to obtain the same reaction rate. This may significantly reduce the use of amino acids, HOBt, and DCC required per gram of product.
A numerical analysis of the diffusion to and through the particle is shown in FIGS. 3 and 4. The concentration of reactants will be highest at the surface of the particle and the concentration of reactants within the particle will increase over time as the reactants diffuse or pass into the resin particle. FIGS. 3 and 4 show that the rate of concentration buildup over time (.tau.) depends on the mass transfer coefficient (H) through the film at the surface of the resin particle and the relative distance zeta =(r/R) through the particle at which the concentration is measured, where R is the overall radius of the particle (particle size) and r is a point measured from the center of the particle. Thus r/R=1 at the surface of the particle and r/R=0 at the center of the particle. Each of the four curves in FIG. 3 represents a concentration profile at a given time .tau. when there is no film resistance (H=.infin.) at the surface of the particle.
FIG. 4 has two plots similar to FIG. 3, but wherein the resistance to mass transfer through the film at the surface of the particle is increased (i.e., H is decreased). By comparing the two plots of FIG. 4 (H=1 and H=3) it can be seen that the concentration within the particle rises more quickly with lower film resistance (i.e., higher H) at the surface of the particle.
FIGS. 3 and 4 show that with significant resistance (both at the surface and in the pores of the particle), the concentration buildup within the particle is slower. This points to a need to reduce both the internal pore resistance and the external surface film resistance so that the mass transfer to the reactants to and through the particle can be increased. Mass transfer of reactants to the resin-bound free amines may be enhanced by increasing the concentration of reactants in the liquid phase, and by decreasing the pore length (using smaller diameter resin particles). In addition, the resistance to mass transfer through the film may be reduced by increasing the velocity of the bulk fluid relative to the resin particles, since film coefficients for spherical bodies are a function of the fluid velocity. A reactor design which addresses these diffusion problems simultaneously is not presently available.
Since the initial development of solid phase peptide synthesis (SPPS) by Merrifield in 1963, a number of innovations have been made in reactor design for peptide synthesis. The earliest reactors were based upon shaken flasks, while later reactor designs included stirred-tank reactors, centrifugal reactors, and tubular reactors. All of these operated with the resin bead suspended or flooded in a liquid phase. Stirred-tank reactors (STR's) are commonly used in peptide synthesis, but they suffer from certain limitations. Because the beads are in suspension, the opportunities to increase the liquid velocity relative to the particles in a stirred-tank reactor are extremely limited. This is because the inertial and viscous forces imposed by the moving liquid upon the suspended particles tend to drag the particles along with the liquid. The fluid velocity field relative to the particles, however, can easily be increased in a packed-bed reactor, in which the resin particles remain fixed, but liquid is forced through the resin bed. However, the bottom frit in both stirred tank and packed bed-reactors required to filter the liquid phase from the resin must be capable of withstanding the pressure differential required for the filtration. As the reactor size is increased, the frit must be fabricated of progressively reinforced materials in order to avoid mechanical failure. On the other hand, if the reactor diameter remains constant, the resin depth on the filter frit increases significantly as the batch size increases. The increased resin depth results in progressively longer drainage and filtration times and exposure times of the resin-bound peptide to the reactants, reactive intermediates, and solvents. For these reasons, neither a stirred-tank reactor with a built-in filter nor a packed bed reactor represent the most efficient type of reactor for a large (multi-kilogram) scale SPPS process.
Although the changes in particle size over the course of the reaction have led to some design difficulties, tubular reactors, such as packed-bed reactors, still offer some advantages over other reactors. Packed bed reactors allow washing to proceed as a displacement operation, rather than a dilution operation as in an STR, provided the amount of "dead volume" between the reactor inlet and the resin bead is minimized. The Reynolds number (Re) in tubular reactors may be very high if the velocity of liquid is high relative to the resin beads. However, high flow rates create high pressures within the reactor, and the high pressures may have a detrimental effect upon the resin. Nevertheless, since mass transfer coefficients increase with increased Reynolds numbers, tubular reactors can have lower liquid film resistance to mass transfer than stirred tank, suspension reactors.
The use of packed beds, however, has its own set of problems. The resin can expand up to 3.times. its original size when washed with DCM and can shrink to 1/3 its original size when washed with methanol. This can amount to a 9.times. change in the overall volume of the resin bed. Elimination or minimization of dead volume therefore is very difficult to accomplish. The packed bed, which has a height much greater than its diameter, has a significant amount of wall surface area which impedes the expansion and contraction of the resin bed. When the resin bed is expanded, it packs against the walls of the vessel and reduces the void space between the resin particles. A high pressure thus is required to push all the wash through the bed in the required amount of time. This high pressure can damage the resin beads, create fine particles which may block the filter, and potentially damage or break the filter frit. If high pressure is not used, then flow through the bed will be too slow and the peptides may be allowed to react for too long and may undergo side reactions. Some amino acids or peptide coupling steps are very sensitive to reaction time. Arginine, for example, may react with itself to form cyclic structures instead of coupling to the peptide chain. Exposure time can therefore become a very important factor.
Several attempts have been made to design a tubular reactor which overcomes the constraints imposed by the resins. These prior attempts include:
(1) Ignoring the problems with resin swelling. Verlunder, et al., U.S. Pat. No. 4,192,798 discloses approaches in which the reactor pressure drop is at least 200 psi, and up to 10,000 psi or more. They claimed quantitative yields, and that reactions which take hours in other reactors were completed in minutes. Difficulties with this type of reactor include degradation of the resin, blockage of the outlet frit, maintaining a uniform axial flow throughout the column upon scale-up, inefficiencies in washing due to dead volume, and the cost of the column and high-pressure pumps. This type of reactor is essentially an industrial-scale HPLC.
(2) Allowing axial expansion of the column. Baru, et al., WO 88/909010.6, SU 4117080 developed a zero dead volume reactor in which one end of the reactor was allowed to float with the resin. A small weight was added to the top piston of the reactor to provide a constant force to the floating head of the reactor. As a result, the column was operated at a low pressure drop to avoid spilling solvent out around the top head, making it very sensitive to blockage in the outlet frit. Accumulation of DCU in the bed or within the frit which may increase the pressure drop and could have safety and environmental consequences. In addition, the low pressure drop constraint requires a low liquid velocity, resulting in low Reynolds numbers and possible maldistribution of the liquid flow. The low liquid rates also result in longer exposure times to the reactants and washes.
(3) Use of gel supported by a rigid polymer. Atherton, et al., JCS Chemical Communications, p. 1151 [1981], developed a rigid polymer in which gel could be contained within the macropores. The gel could swell and shrink, but the volume of the rigid polymer beads would remain constant. As a result, high liquid flow rates, low pressure drops, and constant resin volume could be obtained. However, it is most likely that a reduction of mass transfer will occur with these types of supported resins, since the dffusional path of the reactants would increase after adding film and pore diffusion through the supports. In addition, these types of resins are expensive.
(4) Allowance for dead volume. Lapluye and Poisson, PCT publication no. WO 92/115867 developed a piston-type reactor with fritted ends. In this type of reactor, resin is placed in a hollow piston which is cycled upwards and downwards within a larger cylinder full of solvent and reactants. This type of reactor is essentially similar to a shaker or fluidized bed reactor, and the mixing motion of the piston reactor should compensate for the differences in densities of the resin and the solvents. It appears that the efficiency of the washing should be the same as, or somewhat better than, smaller fluid phase volumes than that in an STR. However, the volume of solvent used relative to the resin mass is probably considerably greater than that required by a typical tubular reactor. The Reynolds numbers are probably quite low, and it may be difficult to scale up the mechanics. In addition, heat can be generated due to "viscous dissipation" effects caused by the motion of the piston through the liquid, requiring some additional means of cooling.
At this point, there does not appear to be a tubular reactor which does not have some serious limitations in cost, safety, or efficiency.
Reactors based upon the design of a rotating bowl have also been developed. Rotating bowls or centrifugal reactors can allow for increasing the liquid velocity relative to the resin particle. Birr, German Patent No. 2,017,351, discloses a "washing machine" reactor in which a porous basket is initially loaded with resin, then spun to a moderate speed while submerged in a liquid. The centrifugal forces cause the resin particles to form a bed on the inside walls of the basket, and cause a moderate degree of fluid recirculation through the resin bed. The bowl is flooded (i.e. filled with liquid), as is the resin bed. The drag imposed upon the basket by the liquid bath imposes high torque upon the drive motor, and will also cause the generation of heat. For these reasons, the rotational speed of the Birr reactor will be relatively slow and the relative fluid to solid velocity will be limited. The limits on rotational speed will almost certainly result in a non-uniform resin bed that is shallow at the top and deep near the bottom. The recirculating liquid will tend to follow the path of least resistance, and thus will "short-circuit" through the shallower part of the bed. As a result, the contact between the reactant rich liquid and resin will not be consistent throughout the reactor. In addition, the lower velocity of the liquid with respect to the resin will result in lower mass transfer through the surface filn surrounding the particle. Finally, the volume of solvent required to submerge the rotating basket will be large compared to the volume needed to soak the resin charge. This will thus also require a significant volume of reactants relative to the resin mass. This large volume will result in the use of a low concentration solution, which consequently results in a slower reaction rate and longer reaction time. It may therefore require a larger amount of amino acid to maintain the amino acid concentration at a level which will result in acceptable reaction rates.
Another type of centrifugal reactor based upon a flooded "hollow rotor" was developed by Anderson and Anderson, U.S. Pat. No. 5,186,824. The liquid flow path in the Anderson et al. reactor is axial rather than radial, and the geometry is irregular for the liquid flow fields. The point at which liquid is introduced depends upon the density of the liquid in relation to the density of the most recently added liquid. Also, little room is provided for expansion of the resin. As a result of expansion and contraction, the exposure of resin to the liquid phase is likely to be non-uniform. Complete and uniform contact and removal of liquid from the rotor may be very difficult to accomplish.